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METHODES
by
SUGIYONO
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INTRODUCTION
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Particle bombardment
b) Electroporation
c) Microinjection
d) Chemical induction
e) Protoplasts fusion
Agrobacterium-mediated transformation
Agroinfiltration
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1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
2. Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and
preventing aging)
Nopaline
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Constructing transgenes
The minimal requirements for the transgene are a
promoter, coding region, and terminator
General structure:
5---Promoter ..Coding region.terminator---3
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1. Constitutive
CaMV 35S: from Cauliflower mosaic virus 35S rRNA
gene
2. Tissue specific
Glutelin GT1: Endosperm specific
3. Inducible
Cis-Jasmone: Arabidopsis genes induced by
stress/wounding
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tumefaciens
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1. Antibiotic resistance
2. Herbicide resistance
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Plasmid map
bar nos
35S gusA-intron
ter
(monocot)
ampR
ColE1 ori
BX EH
35S
35S
crtI
RBR
35S
ter
XhoI
ssu
ter
XBE
XhoI
nptII
BX
XhoI
nos
XBE
XhoI
XB E B
LBR
lox
lox
ColE1 ori
spc/sptr
RK2
pBECKS2000.4
35S gusA-intron
(monocot)
XBE
ampR
35S
ColE1 ori
spc/sptr
RBR
crtI 35
35S
S
ter
lox
ColE1 ori
BX EH
XhoI
hph
BX
XhoI
nos
ter
BE
XhoI
nptII
E XH
XhoI
nos
XBX
nos
LBR
lox
RK2
pBECKS2000.6
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Every induced
Agrobacterium cell
produces one T-strand.
VirD1 and VirD2 are
involved in the initial Tstrand processing,
acting as site-and
strand-specific
endonucleases.
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The T-complex is
composed of at least
three components:
one T-strand DNA
molecule, one VirD2
protein, and around
600 VirE2 proteins.
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2)
4)
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: www.plantmethods.com/content/2/1/16/figure/F1
http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif
Agrobacterium-mediated transformation of
monocotyledons can be achieved with the same efficiency
as dicotyledons
The T-DNA is transferred from Agrobacterium to
dicotyledons and monocotyledons by an identical
molecular mechanism and with similar efficiency
The majority of plants produced by this method are free
of morphological aberrations
The fertility of the transgenic plants was found to be
similar to seed-grown plants, and the segregation ratio for
the transgene was exactly 3 : 1, which supports the
conclusion that the transgene has been integrated into the
genome